mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Analysis of Cryptosporidium hominis genome
table summary.jpg|Figure 2: C. hominis genome compared to C. parvum and P. falciparum genomes. Source: reference (6). All pathways.jpg|Figure 3: Schematic demonstration of select metabolic pathways in C. hominis. Source: reference (6). metabolic pathways.jpg|Figure 4: Comparison of metabolic enzymes of C. hominis and P. falciparum. Source: reference (6). putative drug targets.jpg|Figure 5: Putative drug targets in C. hominis. Source: reference (6). Cryptosporidium hominis is an apicomplexan, intracellular protist that infects the small intestine causing diarrhea (1). Cryptosporidium parvum and Cryptosporidium hominis are the two major species of Cryptosporidium that cause diarrheal disease in humans called Cryptosporidiosis (2). The diarrhea is self-limiting in immunocompetent individuals, but can be fatal in case of immunocompromised people (3). It is also the second leading cause of diarrhea in children under 12 months of age in developing countries (4). The parasite replicates in thr host (human) and excretes thick walled oocysts with fecal matter for transporation to another host (Figure 1). These oocysts are usually transmitted by contaminated water as the oocysts are highly resistant to a lot of the waste water treatments, especially chlorine. There are no effective vaccines and the present available drug, nitazoxanide is not very effective against the pathogen in children and immonocompromised individuals, lending importance to the imperative need to develop therapeutics against Cryptosporidiosis (5). One of the major hindrances has been the lack of tools to study this parasite as there are no established methods to culture it in vitro or to genetically engineer it. Due to these limitations, the genome sequence of C. hominis provides a very useful resource for developing theraupetics against the pathogen (6). Approach C. hominis TU502 strain oocysts were obtained from an infected child from Uganda and propagated in gnotobiotic piglets. The expanded oocysts were then isolated from feces of gnotobiotic piglets and expanded in neonatal calves. Genomic DNA was isolated after surface sterilization of oocysts. Bacterial artificial chromosome (BAC) and shotgun clones were constructed and sequenced to obtain genomic sequence of C. hominis. Analysis of the Genome Overwiew C. hominis has 8 chromosomes containing about 9.2 million base pairs, that is AT rich with G+C content being only 31.7%, which is vey similar to C. parvum genome (Figure 2). C. hominis and C. parvum have a higher percentage of coding regions in their genome compared to P. falciparum. C. hominis ''is predicted to have a relatively lower number of introns, approximately 5-20%. Energy Production An overview of various metabolic pathways in ''C. hominis is shown in Figure 3. C. hominis ''genome codes for a large number of transporters and seems to depend on the host for a large number of nutrients. For energy, it relies mainly on glycolysis and lacks vital enzymes for required for Krebs cycle and β-oxidation of fatty acids (Figure 4). It has both, an anerobic pathway utilizing pyruvate:NADP+ oxidoreductase, and an anerobic pathway using an alternate oxidase. Parts of complex I and III are present, but not complex II and IV. This suggests that adenosine triphosphate (ATP) synthesis does not occur by oxidative phosphorylation, but parts of the machinery are probably present to reoxidize NADH. Along with absence of ATP synthesis, it also lacks mitochondrial DNA sequences. However a double membrane-bound organelle is present. This organelle maintains a protein gradient using cardiolipin and performs some of the mitochondrial functions and is hence termed mitochondria-like organelle (Figure 3). comparison-1-10a.jpg|Figure 6a: Gene Ontology studies between ''C. hominis, P. falciparum and S. cerevisiae. Source: reference (6). comparison-2-10b.jpg|Figure 6b: Gene Ontology studies between C. hominis, P. falciparum and S. cerevisiae. Source: reference (6). Comparison to C. parvum.jpg|Figure 7: Comparison of C. hominis genome with that of C. parvum. Source: reference (6). Biosynthetic pathways C. hominis ''lacks key enzymes required for the production of several key building blocks, including simple sugars, amino acids and nucleotids. Unlike other apicomplexan pathogens, the shikimate pathway to make amino acids are absent in ''C. hominis. It instead has a high abundance of amino acid tramsporters to obtain them from the host. Interestingly, it has enzymes to to make complex sugars like starch and amylopectin from precursors. The parasite also lacks enzymes for nucleotide biosynthesis and relies on the salvage pathway instead (Figure 4). Signalling molecules C. hominis ''seems to have cyclic-AMP mediated signalling pathway as it encodes for adenylate cyclase, cyclic-AMP phosphodiesterase and protein kinase A, but lacks genes for trimeric G protein that's associated with this signalling (Figure 4). It also encodes for phosphatidylinoisitol 3-kinase and phospholipase C indicating the presence of calcium and phosphatidylinositol phosphate mediated regulatory mechanisms. Apicoplast ''C. hominis, like C. parvum, seems to lack an apicoplast (Figure 3), otherwise present in all apicomplexan parasites, believed to have obtained from an algae as a result of secondary endosymbiosis. The loss of an apicoplast seems to have occured after the enulment of the alga that gave rise to the apicoplast. This is mainly due to the presence of plant-like enzymes, example D-glucose-6-phosphate ketol-isomerase and 2-phospho-D-glycerate hydrolase, which may be derived from the anciet alga endosymbionts. Gene Ontology (GO) studies Gene ontology (GO) annotations for C. hominis, Plasmodium falciparum and Saccharomyces cerevisiae were done and compared. GO was surprisingly similar among all these organisms (Figures 6a and 6b). One must note here that GO studies can only be done for already known gene families. Thus, the authors predict that the differences seem between the organisms may be due to un-reported gene families or non-conserved genes. Comparison to C. parvum Comparison of C. parvum and C. hominis genomes show that they are very similar with only 3-5% divergence in sequence. Added on, there seems to be no large insertions, deletions, or rearrangements between the two genomes (Figure 7). This suggests that the major differences between the two species lies in gene regulation and functionally significant polymorphisms in the genes. Conclusion The genome sequence analysis have shed light into several pathways in C. hominis that could be potential drug targets as they seem to be unique to the pathogen and divergent from the host. For example, pathways involving synthesis of complex sugars, nucleotide biosynthesis, energy metablosim, aerobic respiratory chain and many more (Figure 5). References 1. Chen XM, Keithly JS, Paya CV, LaRusso NF (2002) Cryptosporidiosis. N Engl J Med 346(22):1723-1731. http://www.ncbi.nlm.nih.gov/pubmed/12037153 PubMed. 2. Xiao L, Fayer R, Ryan U, Upton SJ (2004) Cryptosporidium taxonomy: recent advances and implications for public health. Clin Microbiol Rev 17(1):72-97. http://www.ncbi.nlm.nih.gov/pubmed/14726456 PubMed. 3. Hunter PR Nichols G (2002) Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin Microbiol Rev 15(1):145-154. http://www.ncbi.nlm.nih.gov/pubmed/11781272 PubMed. 4. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet. 2013;382(9888):209-22. Epub 2013/05/18. http://www.ncbi.nlm.nih.gov/pubmed/23680352 PubMed. 5. Abubakar I, Aliyu SH, Arumugam C, Hunter PR, Usman NK (2007) Prevention and treatment of cryptosporidiosis in immunocompromised patients. Cochrane Database Syst Rev (1):CD004932. http://www.ncbi.nlm.nih.gov/pubmed/17253532 PubMed. 6. Xu P, Widmer G, Wang Y, Ozaki LS, Alves JM, Serrano MG, et al. The genome of Cryptosporidium hominis. Nature. 2004;431(7012):1107-12. Epub 2004/10/29. http://www.ncbi.nlm.nih.gov/pubmed/15510150 PubMed